Kaempferol-loaded bioactive glass-based scaffold for bone tissue engineering: in vitro and in vivo evaluation

Due to the increasing prevalence of bone disorders among people especially in average age, the future of treatments for osseous abnormalities has been illuminated by scaffold-based bone tissue engineering. In this study, in vitro and in vivo properties of 58S bioactive glass-based scaffolds for bone tissue engineering (bare (B.SC), Zein-coated (C.SC), and Zein-coated containing Kaempferol (KC.SC)) were evaluated. This is a follow-up study on our previously published paper, where we synthesized 58S bioactive glass-based scaffolds coated with Kaempferol-loaded Zein biopolymer, and characterized from mostly engineering points of view to find the optimum composition. For this aim, in vitro assessments were done to evaluate the osteogenic capacity and biological features of the scaffolds. In the in vivo section, all types of scaffolds with/without bone marrow-derived stem cells (BMSC) were implanted into rat calvaria bone defects, and potential of bone healing was assessed using imaging, staining, and histomorphometric analyses. It was shown that, Zein-coating covered surface cracks leading to better mechanical properties without negative effect on bioactivity and cell attachment. Also, BMSC differentiation proved that the presence of Kaempferol caused higher calcium deposition, increased alkaline phosphatase activity, bone-specific gene upregulation in vitro. Further, in vivo study confirmed positive effect of BMSC-loaded KC.SC on significant new bone formation resulting in complete bone regeneration. Combining physical properties of coated scaffolds with the osteogenic effect of Kaempferol and BMSCs could represent a new strategy for bone regeneration and provide a more effective approach to repairing critical-sized bone defects.


Results
In this study, we made bare (uncoated, B.SC) and Zein-coated 58S bioglass-based scaffolds without/with Kaempferol loading (C.SC or KC.SC) to offer mechanical augmentation by Zein and osteogenesis by Kaempferol to the scaffolds, and assessed them from biological and osteogenesis point of view in vitro and in vivo. Moreover, the scaffolds were loaded with bone marrow stem cells (BMSCs) to amplify osteogenesis capacity. Figure 1 shows the surface structure, cell attachment, and bioactivity of bare and coated scaffolds. According to scanning electron microscope (SEM) images of scaffolds, Zein coating (7% (w/v)) covered surface and filled micro-cracks without filling surface micropores. Cell attachment experiment proved that MG-63 cells could attach and spread on bare and Zeincoated scaffolds as well. Bioactivity assessment of scaffolds was done through immersion in SBF for 14 days. The SEM images showed hydroxy carbonate apatite (HCA) particles covered bare and Zein-coated scaffold and coating had not negative effect on bioactivity. Also, Zein coating at concentration of 7% (w/v) could improve compressive strength (3.06 MPa) compared with bare scaffold (0.88 MPa) 44 .

Characterization of bone marrow-derived mesenchymal stem cells (BMSCs). Isolated BMSCs
(passage 3) were evaluated by flow cytometry based on specific surface markers whose result reported as Fig. 2ad. Analysis showed that BMSCs were positive for CD90 and CD44 markers and negative for CD34 (endothelial marker) and CD45 (Hematopoietic marker) markers. Figure 2e shows the optical microscopy image of isolated BMSCs.
In vitro assessments of biological activity and gene expression. Calcium deposition assessment. Osteoblast-like differentiated cells started to deposit calcium in their extra cellular matrix (ECM) recognized by alizarin red S staining. After 21-days treatment with conditioned media, deposition of calcium nodules in C.SC and KC.SC was higher than B.SC and the best calcium deposition was occurred because of Kaempferol in KC.SC scaffold (Fig. 3a).
Alkaline phosphatase (ALP) activity. To estimate the early differentiation of BMSCs to osteoblast-like cells, BMSCs cultured in conditioned media and their ALP activity were measured after 7 and 14 days. The result (Fig. 3b) (Fig. 3d).

Scaffold transplantation and in vivo study.
To assess osteogenesis and bone regeneration, criticalsized defects were created in the rat calvarias and various types of scaffolds with or without BMSCs were trans-   www.nature.com/scientificreports/ planted. Afterwards, regeneration potential was investigated using radiography, µCT, and cell staining after 4 and 12 weeks. Figure 4 shows radiographic images of the rat calvarias 4 and 12 weeks post-implantation. Considering radio-opacity caused by mineral deposition, bone regeneration was not observed in the control group. All 58S-based scaffolds especially cellloaded ones could improve bone formation after 4 and 12 weeks and BMSC presence had great influence on scaffold-bone integration. The µCT analysis (Fig. 5) confirmed the capacity of various scaffolds for bone formation especially at the edge of calvaria by cell-loaded C.SC and KC.SC scaffolds.

Radiological and three-dimensional micro-computed tomography (3D μCT).
Histomorphometric analysis. Figure 6 shows quantitative histomorphometric analysis including percentages of fibrous connective tissue (FCT%, Fig. 6a), new bone area (NB%, Fig. 6b), and number of cells and osteons ( Fig. 6c). After evaluation periods (4 and 12 weeks), although all 58S-based scaffolds could improve bone formation compared with the control group, it was obvious that BMSC-loaded KC.SC had the best effect whose NB% increased by ~ 200% after 12 weeks. However, almost all scaffolds could prevent formation of fibrous connective tissue and BMSC-loaded KC.SC had the lowest FCT% among all investigated groups. As seen in Fig. 6c, the number of osteoblasts, osteocytes, and osteons were the highest in KC.SC + cell groups compared with other groups after 12 weeks. On the contrary, the highest number of fibroblasts belonged to the control group.
Immunohistochemical analysis. Figures 7 and 8 include H&E, MT, and IHC results after 4 and 12 weeks postimplantation, respectively. The H&E staining showed that fibrous connective tissue (FCT) filled defect and new bone formation was insignificant in the control group. For defects implanted by B.SC, B.SC + cell, C.SC, and C.SC + cell groups, the implantation sites were substituted with FCT, multinucleated giant cells (MGCs) were visible, and bone reconstructed partly after four weeks post-surgery. In contrast, new bone was formed in KC.SCimplanted groups (with or without BMSC) after 4 and 12 weeks. Despite incomplete osteointegration after four weeks, new bone formation raised after 12 weeks, the defect implanted by KC.SC + cell group was filled entirely by new bone, and bone maturation associated with complete osteointegration occurred after 12-weeks implantation. Immunohistochemical staining of tissue sections for the OCN and OPN markers confirmed that there were not any differences in expression of OCN and OPN markers between B.SC, B.SC + cell, and control groups. Also, OCN and OPN deposition in C.SC, C.SC + cell, and KC.SC groups were considerably higher than other treatments and control groups. In the KC.SC + cell group, bony islands were observed across the defects. respectively. An asterisk indicates the statistically significant difference between genes and control as measured by Student's t-test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Discussion
Since the bone injuries larger than critical size defect cannot be reconstructed, it is important to provide constructs with suitable function for bone regeneration. Optimal physical characteristics, biocompatibility, biodegradation, osteogenesis, and osteointegration are essential features for bone tissue engineering. Also, stem cells, accompanied by biomaterials or tissue-engineered constructs, could improve regeneration of critical-sized bone defects 5,7 . In the current study, we synthesized BMSC-seeded bioglass scaffolds with/without Kaempferol-loaded Zein coating and evaluated them for bone tissue engineering in vitro and in vivo.
Although coating the scaffold with a polymer solution such as Zein would cover the surface and pores, this new structure with the pore size ranging from 200 to 500 µm would be appropriate for cell migration inside the scaffolds [50][51][52] . Meanwhile, the mechanical strength of coated scaffolds was threefold higher than that of uncoated scaffolds which was parallel to other published reports 27,28,53,54 . Bioactivity analysis showed that HCA crystals covered the surfaces of all scaffolds confirming the stimulating effect of Zein coating on bioactivity due to the presence of proper chemical groups as nucleation sites for HCA deposition 27,28,55 . Besides, attachment and www.nature.com/scientificreports/ proliferation of BMSC cells on Zein-coated and bare scaffolds proved that Zein hydrophobicity did not have a negative effect on cell functionality 34,56 .
The BMSCs are good cell sources for bone regeneration because of their self-renewal and multipotential differentiation capacity 57 . They are positive for representative expression markers such as CD29, CD90, and CD44, whereas they are negative for hematopoietic markers including CD45, CD11, and CD34 58 .  www.nature.com/scientificreports/ Alkaline phosphatase (ALP) is a metalloenzyme that encodes various genes in different tissues. ALP has serine phosphate in its active site and can react with O-H in alkaline pH leading to the release of inorganic phosphate 59 . ALP activity is known as an osteogenesis indicator, and early marker of the osteoblast differentiation 35 . The MSCs exude ALP in the early stage of osteo-differentiation and has a positive effect on the beginning of calcification and formation of calcium phosphate clusters by interacting free phosphate with Ca 2+60,61 . Our results confirmed the release of ALP increased in all experimental groups, especially KC.SC after 7-and 14-days treatment confirming osteo-differentiation onset in all groups. Further, ALP is a differentiation characteristic whose higher  Zein as a biocompatible and biodegradable polymer has osteogenic capability 5 , and can improve bone formation 35,38 . By having no negative effect on the surface adhesion and proliferation of mesenchymal stem cells (MSCs), its osteoconductive properties stimulates MSCs to differentiate towards osteoblasts in the presence of dexamethasone 34 . Zein can enhance the expressions of osteogenic genes such as ALP, COL1 and OCN 36 . In vitro studies about Kaempferol action on osteogenesis have proved that it causes upregulations of various specific genes such as ALP, COL1, OCN, OSX (osterix), BMP-2 (bone morphogenetic protei-2), OPN (osteopoetin), induces mineralization of osteoblasts and calcium deposition, and provokes formation of bone nodules. It stimulates the differentiation murine preosteoblastic MC3T3-E1 cells, and improves their ALP activity and calcification. Bone formation occures through the intramembranous or endochondral ossifications during which MSCs directly or indirectly differentiate into osteoblasts to form finally mineralized bone matrix. Kaempferol can modulate different matrix metalloproteinases to commit MSCs to the osteoblastic lineage by upregulating ALP, Runx-2, OSX and OCN, and downregulating PPAR-γ to prevent adipogenesis 41 . According to Adhikary et al., the osteogenic action of Kaempferol is owed to the BMP-2 upregulation resulting in osteoblast proliferation, and exhibiting high expressions of ALP, OSX, OCN, Runx-2, and COL1 in dexamethasone-induced rat calvarial osteoblasts 65 .
Three-dimensional µCT presented more osteogenic activity for cell-loaded scaffolds compared with cell-free scaffolds. Although all scaffolds with or without BMSC were osteoconductive and osteoinductive, new bone regeneration was significant in the center and sides of defects filled with KC.SC + cell after 12 weeks agreeing with in vitro experiments. In bone repair, biomaterials act as a platform to support stem cell adhesion and proliferation. BMSCs when exposed to chemical compounds, physical stimulations, and surface of biomaterials differentiate into osteogenic cells by releasing matrix components promoting calcification, and forming new bone. Meanwhile, growth factors such as BMP-2 are secreted from committed cells and have a positive effect on attracting, proliferating, and differentiating of mesenchymal progenitor cells resulting in formation of bone tissue in vivo 66 . Our results were parallel with the study accomplished by Mazaki et al. in which bone mineral density of lumbar spine increased by Kaempferol treatment 67 . Moreover, As explained previously 61,68 , periosteal osteoprogenitor cells invade the defect site and cause new bone formation at the peripheral area and on the surface of scaffolds.
As reported earlier 69 , there is a direct correlation between the number of osteoblasts and new bone formation. The histomorphometric analysis showed that osteoblasts, osteons, and osteocytes in KC.SC + cell group were more than other groups after 12 weeks post-surgery. And, new bone formation was the highest and the fibrous connective tissue was the lowest in KC.SC + cell group after 12 weeks.
Monocyte progenitors could differentiate into multinucleated giant cells (MGCs) whose occurrence is enhanced after bone failure, chronic inflammation, tumor formation, and granulomatous disease. Macrophage is an important type of MGCs that is produced in response to infection. Histopathology results showed that MGCs could adhere to scaffold surface and contribute to inflammation and regeneration 70 . Scaffold degradation depends on macrophages' activity that results in polymer degradation and ion release providing a suitable environment for new bone formation. Further, infiltration of macrophages into the implantation site can cause an inflammatory reaction 70,71 . In the current study, defect sites implanted by all experimental groups with or without BMSCs underwent FCT and MGCs accumulation after four weeks post-implantation that could be a symptom of implant-related inflammation. However, macrophages were not seen in KC.SC + cell group after 12 weeks indicating termination of inflammatory process at this time. It could be concluded that implant degraded and osteointegration and remodeling progressed after 12 weeks post-implantation 72 . In the control group, FCT filled the defects and new bone formation was negligible. New bone formation increased in KC.SC but fibrous tissue between the implant and host bone showed that osteointegration was incomplete at both time points 73 . Based on our findings, BMSCs and Kaempferol as an osteogenic agent in KC.SC + cell group could improve new bone formation and osteointegration compared with other groups.
Apart from differentiation capacity into different cell lineages, research findings present that mesenchymal stem cells display broad immunomodulatory properties and can suppress immune rejection 5,7 . So, better bone formation in BMSC-loaded scaffolds might be caused by immunological and biological roles of BMSCs. Aligned with Tu et al. 5 , administration of BMSCs not only supply osteogenic cell source for bone regeneration but also provide growth factors having a positive effect on differentiation and new bone formation.
The immunohistochemical assessment did not show any differences in expression of OCN and OPN markers between B.SC, B.SC + cell, and control groups. However, the number of OPN-and OCN-positive cells in C.SC, C.SC + cell, and KC.SC was considerably higher than expressions of those genes in the control group. It is believed that OCN produced by osteoblasts is needed for mineralization and calcium ion homeostasis 74 . OCN stimulates the maturation and migration of osteoclast precursors, and contributes to the formation and maturation of hydroxyapatite crystals. It is not expressed in osteoblasts at the very early stage of the mineralization process, and detected in the bone matrix after significantly progressed mineralization. Due to very low expression in the early stages of osteoblast differentiation, OCN is considered as a marker of mature osteoblasts 75 . In the C.SC, C.SC + cell, and KC.SC groups, the OCN-positive cells spread throughout all the regions homogeneously showing formation of mineralized ECM. Mature bone tissue and osteon formation in KC.SC + cell group was aligned with histomorphometric analysis; therefore, osteogenesis process finished, OCN decreased, and bone islands formed.

Conclusion
In this study, the osteogenic potential of porous 58S-based scaffolds including bare, Zein-coated, and Kaempferolloaded scaffolds was investigated in vitro and in vivo. According to in vitro and in vivo findings, 58S-based scaffolds coated with Kaempferol-loaded Zein with BMSCs could promote osteogenesis resulting in better ALP activity, bone-specific gene expression, and bony island formation. So, combining physical properties of coated scaffolds with the osteogenic effect of Kaempferol and BMSCs may represent a new strategy for bone regeneration and provide a more effective approach to repairing critical-sized bone defects.

Materials and methods
All methods were carried out in accordance with relevant guidelines and regulations.
Scaffold preparation and engineering characterization. The protocols for the synthesis of 58S bioactive glass, scaffold preparation and characterization were reported in our previous article 44 . Briefly, the 58S bioglass was synthesized by mixing tetraethyl orthosilicate (TEOS, 39.2% (v/v)), triethyl phosphate (TEP, 1.7% (v/v)), and calcium nitrate tetrahydrate (Ca(NO 3 ) 2 ⋅4H 2 O, 26.4% (w/v)) in H 2 O. After keeping three days at 25 °C for gelation, the mixture was aged at 60 °C, and dried at 120 °C for 24 h. The prepared 58S bioactive glass was characterized by differential thermal analysis (DTA), thermogravimetric analysis (TGA), X-ray diffraction (XRD), and Fourier-transformed infrared spectroscopy (FTIR). Then bioactive scaffold was made by immersing polyurethane foam into aqueous slurry of 58S (40% (w/v))-poly vinyl alcohol (PVA, 5% (w/v)), followed by heating at 350 °C for 30 min to burn the foam and 1250 °C for three hours to sinter the scaffold body. In the following, prepared scaffold was coated with Zein solution to increase mechanical strength, and loaded with Kaempferol to improve its osteogenic activity. Full structural, surface morphology and mechanical evaluation, assessment of bioactivity and degradation, drug release, and surface cell attachment were carried out. In current study, finally approved 58S scaffold coated with Zein-Kaempferol (7% (w/v)-400 µM) was used for in vitro and in vivo studies. In vitro study. To evaluate the osteogenic capacity, biological features of the scaffolds including bare scaffolds (B.SC), Zein-coated scaffolds (C.SC), and Kaempferol-loaded C.SC (KC.SC) were assessed by analyzing specific enzymatic activity, staining deposited minerals by cells, and measuring gene expression.

BMSCs extraction and characterization. To isolate BMSC cells, rats
Calcium deposition assessment. Alizarin red staining was accomplished to detect calcium deposition in the extracellular matrix after 21 days. Because of the presence of calcium in scaffolds, it was difficult to distinguish calcium deposition in ECM. Thus, BMSCs were cultured in the plate, and conditioned media (10% FBS-supplemented DMEM containing 0.1 mg/mL of each scaffold) were added. After 21 days, media was removed; the BMSCs were washed with PBS, and fixed with 4% paraformaldehyde (PFA) for 40 min at room temperature. Then, PFA was pulled out and replaced with alizarin red solution (2% (w/v) in deionized water, pH 4. 1-4.3). After incubation at 4 °C for 40 min, the stained BMSCs were observed under an inverted microscope (Labomed, USA).
Alkaline phosphatase (ALP) activity. Colorimetric ALP assay kit (Abcam, USA) was applied to measure the alkaline phosphatase activity according to the manufacturer's instruction. Briefly, supernatants of the BMSCs cultured on the different scaffolds were accumulated every two days for 7 and 14 days. Then, 80 µL of supernatant was incubated with 50 µL of p-nitrophenyl phosphate (pNPP, Sigma-Aldrich, USA) at 37 °C for one hour in darkness, and absorbance was measured by a microplate reader (BioTek, USA) at 405 nm.
RNA isolation and gene expression evaluation. After treating BMSCs with conditioned media of different scaffolds for 14 and 21 days, BMSCs were washed by PBS, trypsinized, and total RNA was extracted using RNA extraction kit (Yekta Tajhiz, Iran). RNA integrities and purities were estimated using a Nanodrop spectrophotometer (Thermo scientific, USA) by considering the ratios of 260/280 and 260/230 about 2 as purity and integrity criteria. Complementary DNA (cDNA) was synthesized using RT-PCR Pre-Mix BioFact kit (BIOFACT, South Korea), and amplified by Corbett Rotor-Gene 6000 (Qiagen, UK) using HOT FIREPol EvaGreen qPCR Dawley rats with an average weight of 250-300 g were used in this experiment. Scaffolds were divided into six groups: bare scaffold (B.SC), cell-seeded B.SC (B.SC + cell), Zein-coated scaffold (C.SC), cell-seeded C.SC (C.SC + cell), Kaempferol-loaded C.SC (KC.SC), and cell-seeded KC.SC (KC.SC + cell). Two days before surgery, the total BMSC numbers of 5 × 10 5 was seeded on each sample. The rats were anesthetized, shaved, and sterilized for surgery, and a circular defect (diameter of 8.0 mm) was created in calvarium under steady DPBS (Dulbecco's phosphate-buffered saline) pouring to prevent overheating. Then the scaffolds were implanted in the calvarium defects. To prevent infection, enrofloxacin 5% was injected into rats before surgery, and added to drink water (0.5% (v/v)) three days after surgery. Considering calvarium defect without any scaffold as control group, ten rats were observed in each experimental group for 4 and 12 weeks (five rats for each time point).
Radiological and three-dimensional micro-computed tomography (3D µCT) evaluation. After 4 and 12 weeks post-implantations, animals in each group (five rats each time point) were sacrificed by overdosed inhalation of carbon dioxide. Tissue samples were collected by preserving surrounded calvarium bone. Primary evaluation of bone regeneration was conducted by digital radiography (Kodak direct view CR850, Japan) immediately. The 12-weeks related samples were fixed by formalin (10%, pH 7.26) and 3D µCT was performed to analyze 3D bone regeneration using a desktop 3D µCT instrument (LOTUS-NDT, Behin Negareh.Co, Iran). Samples were sealed at a micro-focus X-ray tube, and scanning was performed by setting suitable parameters (voltage of 80 kV, current of 80 µA, best time of 2 h) resulting in resolution of about 10 µm.
Tissue processing and staining. The collected and fixed tissue samples were decalcified with 14% ethylenediamine tetra acetic acid (EDTA) solution for four weeks. Then, they were dehydrated in different grades of ethanol solutions (70, 90, and 100%), cleared with xylene, embedded in paraffin, and cut into sections with thickness of 5 μm using microtome. All sections were fixed on histological slides, and stained with hematoxylin-eosin (H&E) and Masson's trichrome (MT). The stained samples were evaluated and imaged using an Olympus BX51 light microscope (Olympus, Japan).
Histomorphometric analysis. Histomorphometric analyses of random H&E images were conducted by computer software Image-Pro Plus® V.6 (Media Cybernetics, USA) to quantify the percentage of new bone (NB) and fibrous connective tissue (FCT) in the defect area using Eq. (1): Whole defect were observed by microscope to check new bone formation and the FCT presence. To measure the numbers of cells (fibroblasts, osteoblasts, fibrocytes, osteoclasts, osteocytes), and osteons, four microscopic spans (at 400× magnification) were randomly selected at the margin and center of the defect 78 . For avoiding bias, the samples' areas were chose and microscopic images were taken by a person who did not know anything about the study.
Immunohistochemical analysis. The histological slides of different groups were analyzed for expression of osteocalcin and osteopontin antibodies. The slides were incubated in citrate buffer solution at 60 °C overnight, and blocked with 1% hydrogen peroxide/methanol at room temperature for 30 min. Then, the slides were incubated with primary anti-osteocalcin and anti-osteopontin antibodies (Abcam, USA) at 4 °C for 12 h. The color reaction was developed with ready to use 3,3′-diaminobenzidine (liquid DAB color solution, Dako, Denmark), and the slides were counterstained with 1% (w/v) hematoxylin (Sigma-Aldrich, USA).